1. Field of the Invention
This invention relates to a method and device for making measurements on the surface of a substrate, in particular, to a method and device for making measurements for portions to be measured on a substrate using scan operation (scanning).
2. Related Background Art
In recent years, with the development of biotechnology, there have been increasing needs for making measurements for and detection of the samples from living organisms. The samples from living organisms are, in general, often hard to carry out a close measurement, unlike chemical substances obtained using the technique of synthetic chemistry, because
1. they has extremely wide variegation;
2. their absolute quantity is small; and
3. those having similar physical properties must be distinguished.
To date there have been devised various methods of making measurements for such samples; and among them, the measurements using a solid phase substrate are receiving most attention now. The measuring technique using a solid phase substrate is, for example, such that it allows detection probes (for example, antibody) fixed on or absorbed into a substrate to react with a sample labeled with a fluorescent substance (for example, antigen) on the substrate and makes measurements for the sample by observing the fluorescence on the substrate.
The principal reasons (advantages) that this measuring technique attracts particular attention are, for example, as follows:
1. measurements can be performed for an infinitely small amount of sample by decreasing the amount (area) of the detection probes attached on the substrate, and hence, the absolute quantity of the sample is not required to be large;
2. measurements of multiple items can be performed simultaneously by arraying various detection substances on the substrate; and
3. the substrate is easy to handle because it is not in a liquid phase, but in a solid phase.
The measurements for a sample using a solid-phase substrate have been applied, for example, to the detection of the base sequences of nucleic acids. In such a detection operation, first, various types of single-stranded DNAs (DNA probes) with various base sequences are attached on a substrate in an array and then a DNA labeled with, for example, fluorescent pigment is applied thereto. When there exists in the sample a sequence complementary to the DNA probe on the substrate, the fluorescent substance is adsorbed on the substrate (hybridization); thus, the base sequence contained in the sample can be examined by checking the state of hybridization against the DNA probe on the substrate. As a matter of fact, Affymertrix in the U.S has developed a DNA chip such that as many as about 10,000 types of DNA probes are arrayed on its micro-area by the photolithographic process and has applied the same to the analysis of DNA base sequences.
An area on which each probe is attached in an array consists of extreme-micro-areas. The reason for this is that, when the absolute quantity of the sample is small, a highly sensitive detection is made possible by concentrating the sample on a micro-probe-area so as to allow the amount of sample per unit to be larger, that is, to provide higher sample density.
Scattering the sample on a wide-probe-area lower a detection sensitivity.
Accordingly, detectors for use in the observation of such arrays are often microscopes or devices including a cofocal optical system. In such cases, the whole of the array is not subjected to analyzing and measuring processing, but it needs to be divided into micro-areas and subjected to read processing area by area with a microscope with high magnification. In other words, detectors involving scan operation are needed. Scanning methods include, for example, methods to perform scan operation by moving a light detecting portion over a substrate having certain dimensions and by moving a substrate while fixing a light detecting portion. Thus various types detectors have been designed so as to match various types scanning methods.
For example, an exclusive detector is commercially available from Hewlet Packard which is a device for making measurements for the DNA microarrays by Affymetrix, and this device consists of both a cofocal optical system and a scanner.
In scanners in common use, for example when the substrate to be measured is rectangular, read operation is performed by scanning from one end of the substrate linearly in a fixed direction to the other end and then returning so as to scan the next area, and repeating this motion. In other words, the scan operation involves a reciprocating motion in terms of its scanning direction (scan operation is not performed in one direction, but involves a returning motion).
However, scan operation involving a reciprocating motion gives rise to problems of making the control and construction of the device more complicated and requiring a longer measuring period. The longer measuring period gives rise to another problem of creating a difference in measuring conditions between the areas of the first measurement and the last measurement. In order to prevent this problem, a special device needs to be installed. Further, detection is performed using fluorescent pigments, the problems of color fading and denaturation may occur depending on the pigment.
A discontinuous scan operation involving a returning motion attendant to a reciprocating motion is likely to cause a decrease in positional accuracy compared with a continuous scan operation. This may affect the measurements on the substrate consisting of high-density microarrays.
Accordingly, the object of the present invention is to solve the problems attendant to the above scanning method.
Specifically, an object of the present invention is to provide a method of making measurements for the sample on the measuring surface of the substrate, comprising forming a circular orbit of detection areas on the measuring surface of the substrate by moving the area to be detected with a detector relative to the substrate.
One example of the measuring methods described above is such that the measurements for the sample are made while forming a rotational surface of the subject and the circular orbit as described above by rotating the substrate around the axis perpendicular to the surface of the substrate on which measurements are made, in particular, the measurements are made by moving the detection area relative to the rotational surface of the substrate on which measurements are made.
Another example of the measuring methods described above is such that it performs measurements while forming the circular orbit of the measuring areas as described above by allowing the detector to perform a rotational motion.
The sample described above is fixed on, adsorbed into, or trapped in the substrate as described above and it is, for example, specifically fixed on the substrate by the probes arranged on the surface of the same.
Preferably, the probes and the sample on the substrate as described above are DNA, protein and peptide nucleic acid (PNA). The measurements for the above sample are made using luminescence such as fluorescence and chemicalluminescence.
Specifically, when making measurements for the sample, any one of the absorption, transmission and reflection of the incident light to said sample is measured.
When multiple labels are detected with multiple detectors in the measurements for the above sample, the multiple labels can be detected with their corresponding detectors simultaneously.
Another object of the present invention is to provide a device for making measurements for a sample on the measuring surface of the substrate, comprising:
a detector for measuring for a label from the sample;
a means for supporting the substrate having the sample as the subject of measurements on its measuring surface; and
a means for forming a circular orbit of detection areas on the measuring surface of the substrate by moving the detection areas, in which detection is performed with a detector, relative to the substrate.